Genetic studies in Saccharomyces cerevisiae have indicated that DNA double-strand breaks are the progenitor of most types of homologous recombination events. The DNA double-strand breaks are subjected to exonucleolytic processing, which results in the formation of 3' single-strand DNA tails of about 600 bases in length. Nucleation of the recombination proteins onto these single-strand tails renders them recombinogenic, leading to a search for the chromosomal homolog and the stable pairing of the single-strand tails with the homolog to form heteroduplex DNA. In the later stages, DNA crossover structures called Holliday junctions are generated, and processing of the Holliday junctions gives rise to crossover recombinants. Extensive genetic evidence indicates that heteroduplex DNA formation is mediated by the evolutionarily conserved genes of the RAD52 epistasis group, of which RAD54 and RDH54 are key members. Studies on meiotic recombination have revealed that MSH4 and MSH5 gene products are specifically required for the occurrence of crossover recombinants. Currently, there is a paucity of information on the functions of the Rad54, Rdh54, Msh4, and Msh5 proteins in the formation and processing of recombination intermediates. Rad54 and Rdh54 proteins have been purified to near homogeneity from yeast cells and both have been found to possess a strong DNA- dependent ATPase activity. Rad54 protein exerts a dramatic stimulation on the Rad51-mediates homologous DNA pairing reaction that generates heteroduplex DNA. Further studies are proposed to delineate the action mechanism of Rad54 and Rdh54 proteins in heteroduplex DNA formation, to examine their interactions with recombination intermediates and with other recombination proteins, to purify putative higher order complexes containing Rad54 and Rdh54 proteins, and to investigate the role of Rad54 and Rdh54 proteins in chromatin remodeling. The Msh4 and Msh5 proteins will be purified and biochemical studies will be carried out to test the hypothesis that they interact with the Holliday structure and with other recombination intermediates.